The project innovatively extends nicotine pharmacokinetics to the subcellular level. This is important because nicotine enters neurons, is then thought to enter the endoplasmic reticulum (ER), and then acts as a pharmacological chaperone for several types of nicotinic acetylcholine receptors (nAChRs). The downstream effects are ?inside-out? pharmacology?. Inside-out effects are thought to lead to some aspects of nicotine dependence, as well as neuroprotection by nicotine. However, three key questions remain. We have developed innovative tools for this study. iNicSnFR1 and iNicSnFR2 are genetically encoded, ?intensity- based nicotine-sensing fluorescent reporters?. In test tubes, at nicotine concentrations near those in the brain of a smoker, iNicSnFRs detect nicotine within < 1 s. Preliminary data in cells transfected with iNicSnFRs answer part of Question 1, affirmatively: nicotine enters the ER within a few s after nicotine is applied near an isolated cell, then leaves within a few s after nicotine is washed away. We use adeno-associated viral vectors that express iNicSnFRs. Question (1). There has been no direct proof for the central aspects of inside-out pharmacology: that nicotine enters the ER, that nicotine binds to nAChRs in the ER, and that this binding produces transitions to more tightly binding, chaperoned states of nAChRs. Aim 1 addresses Question 1, by continuing to develop and apply the iNicSnFR tools quantitatively, in mammalian clonal cell lines and in cultured neurons. We will verify the selective targeting of iNicSnFRs to either the ER or the plasma membrane (PM), using membrane-permeant vs impermeant compounds. We then employ iNicSnFR signals in the ER to measure kinetic buffering of nicotine, when it binds to heterologously expressed nAChRs in the ER. Multiple controls and comparisons are used to modify this binding by changing the subcellular localization of nAChRs, their binding affinity for nicotine, and the extent to which they undergo transitions to higher affinity. We also attempt to measure kinetic buffering by endogenously expressed nAChRs, in cultured dopaminergic neurons. We then image the localization of the iNicSnFR signals at greater resolution, to ask whether buffering occurs at sub-regions of ER. Question (2). The time course of nicotine entry and exit from various brain compartments after a pulse of nicotine (for instance, during smoking) is not known. Aim 2 addresses Question 2 by studying the iNicSnFRs expressed in mice. We study iNicSnFR in brain slices from these mice, to provide additional evidence of intended localization. We also study live mice. After a pulse of nicotine is introduced by peripheral injection, we measure the extent and time course of nicotine concentration in the ER. The experiment employs fiber photometry. We will compare these measurements with those for PM-localized iNicSnFR constructs, and with conventional measurements of nicotine in blood and CSF. Question (3). After nicotine accumulates in the acidic lumen of synaptic vesicles by the expected factor approaching ~ 100, is nicotine then released by presynaptic impulses? Aim 3 addresses Question 3. In the high- risk spirit of an R21, answering Question 3 is not on the project's critical path to success, but would enhance the tools' usefulness. Using the brain slice preparation, we ask whether iNicSnFR can detect when presynaptic stimulation releases nicotine from synaptic vesicles. Note that this accumulation and release is not thought to be restricted to cholinergic synaptic vesicles. Answering Questions 1, 2, and 3 prepares for additional research. After decades of uncertainty, we will finally have the answer to the question, which nicotine dose and time course should be used in testing the details of inside-out nicotine action? The measurements from mice can be extrapolated, using standard allometric assumptions, to the brains of smokers or vapers.